1. Volume 37, Issue 6, Pages 971-985 (December 2012)
The DNA Damage- and Transcription-Associated Protein Paxip1 Controls Thymocyte Development and Emigration 
Elsa Callen, Robert B. Faryabi, Megan Luckey, Bingtao Hao, Jeremy A. Daniel, Wenjing Yang, Hong-Wei Sun, Greg Dressler, Weiqun Peng, Hongbo Chi, Kai Ge, Michael S. Krangel, Jung-Hyun Park, André Nussenzweig 
Immunity 
Volume 37, Issue 6, Pages (December 2012)
DOI: /j.immuni
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2. Immunity 2012 37, 971-985DOI: (10.1016/j.immuni.2012.10.007)
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3. Figure 1 PAXIP1 Regulates Thymocyte Development
(A) Flow cytometric analysis of WT and Paxip1−/− thymocytes stained with antibodies against CD4 and CD8.
(B) Graphs showing numbers of total, DPs, CD4+, and CD8+ thymocytes from WT (black) and Paxip1−/− (red) mice. Average number in each population is indicated by the dashed line.
(C) TCRβ, Qa2, and HSA expression in CD4+ and CD8+ cells from WT (black) and Paxip1−/− thymocytes. Same number of total thymocytes from WT and Paxip1−/− were collected for flow cytometry.
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4. Figure 2
Decreased Number of Peripheral T Cells in the Absence of PAXIP1
(A) Left panel, flow cytometric analysis of WT and Paxip1−/− lymph node shows a decreased frequency of TCRβ+, CD4+, and CD8+ SP cells in Paxip1−/− thymocytes. Right panel, bar graph shows the average number of T cells in WT and Paxip1−/− lymph node (n = 16 mice).
(B) Frequency of CD4+ and CD8+ T cells in spleen (left) and percentage of CD19+ and CD3+ lymphocytes in blood respectively was detected by flow cytometry. Representative examples are shown. Bar graphs (bottom) show the average number of CD4+ and CD8+ SP T cells in spleen (n = 9 mice) and CD19+ and CD3+ cells in blood (n = 5 mice) from WT and Paxip1−/− mice. Error bars represent SD.
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5. Figure 3 PAXIP1 Regulates Tcra Recombination
(A) Loss of PAXIP1 decreases H3K4me3 at 3′ Jα segments in DP T cells. Gene tracks represent position of H3K4me3 across a region spanning Jα segments in WT (top) and Paxip1−/− (bottom). The x axis represents the linear sequence of genomic DNA, and the y axis represents the total number of mapped reads per million of mapped reads in 200 nucleotide windows. The genomic scale in kilobases (kb) is indicated above each track.
(B) Jα usage in Paxip1−/− DP thymocytes relative to WT normalized to 1 for each Jα (mean ± SEM of two independent experiments).
(C) Expression of TCR Cα transcripts in WT and Paxip1−/− DP T cells (mean ± SEM of two independent experiments).
(D) TRAV12 (Vα8)-Jα coding joints in Paxip1−/− DP thymocyte genomic DNA relative to WT normalized to 1 for each Jα (mean ± SEM of two independent experiments).
(E) DSBs were detected in serial 3-fold dilutions of genomic DNA isolated from WT and Paxip1−/− DP thymocytes and were detected by Southern blot. Results of two independent experiments are shown.
(F) H3K4me3 ChIPseq profiles of the Tcra locus from Rag2−/− × TCRβ × Paxip1f/f x Lck Cre+ and Rag2−/− × TCRβ x Paxip1f/f × Lck Cre− DP T cells show decreased tag counts in absence of PAXIP1.
(G) Germline transcript abundance in Rag2−/− Paxip1−/− compared with Rag2−/− DPs as measured by real-time PCR.
(H) Genomic instability analysis of metaphase spreads from thymocytes stimulated with anti-CD28 and anti-TCRβ for 2 days. Aberrations associated with the TCR Cα locus at chromosome 14 are represented in black. General instability outside the TCR Cα locus is represented in gray. Schematic and representative images of normal and broken chromosome 14 at the TCRαδ locus are shown. Arrow points to TCRαδ locus (green), telomere is shown in red, and DAPI counterstain is in blue.
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6. Figure 4
PAXIP1 Is Essential for NK T Cell Development and Differentiation
(A) Top panel shows flow cytometry of WT and Paxip1−/− thymocytes stained with PBS-57 loaded CD1d-tetramer (CD1 dTet+) and anti-CD24 (HSA). Bottom panel shows CD44 and NK1.1 expression on gated HSAloCD1 dTet+ thymocytes.
(B) Splenocytes stained with CD1 dTet+ and anti-TCRβ.
(C) Quantitation of NK T cell numbers in thymus (n = 4 mice analyzed for each genotype).
(D) qRT-PCR analysis of Vα14-Jα18 rearrangements in WT and Paxip1−/− thymocytes.
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7. Figure 5
PAXIP1 Dependent H3K4me3 at Promoters during Thymocyte Development
(A) Differences in the distribution of H3K4me3 at promoters in DP and SP T cells. The fold change in H3K4me3 normalized tags per million reads (TPM) at the promoter of each gene in NCBI Build 37 RefSeq is indicated by color (left). The subset of genes with more than 3-fold change in H3K4me3 TPM is magnified (right), and representative genes for six identified categories are enumerated next to the color side bar (right).
(B) PAXIP1 deficiency contributes to deregulation of H3K4me3 in less than 0.3% of genes. The count of gene symbols unaffected (gray), and positively (red) or negatively (blue) regulated by PAXIP1 is shown for CD4+ SP, CD8+ SP, and DP T cells. Gene is designated as PAXIP1-dependent when H3K4me3 TPM at the promoter is changed by at least 3-fold.
(C and D) Gain of H3K4me3 at promoters during WT DP to SP T cell development correlates with deficit in H3K4me3 in the absence of PAXIP1 in SP T cells. Scatterplot of CD4+ SP T cell and CD8+ SP T cell(C and D, respectively) show PAXIP1 -dependent H3K4me3 TMP fold enrichment (y axis) versus difference in H3K4me3 TPM between DP WT and CD4+ SP WT or CD8+ SP WT T cells (C and D, respectively) (x axis). Each point depicts a gene’s promoter in NCBI Build 37 RefSeq. Genes with more than 3 times reduction or increase in SP Paxip1−/− versus SP WT T cells are depicted in red or green, respectively. The 3-fold enrichment boundary is marked by dashed line. Promoter is defined as +/− 5Kb from TSS.
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8. Figure 6 ChIP-seq Analysis of PAXIP1-Binding Sites
(A) PAXIP1-binding sites are enriched for Pax and Zbtb16 motifs. Enriched motif models at PAXIP1-bound sites were identified and computed relative to two background models as described in the methods. The motif of PAX paired-homeodomains was computed based on de novo motif analysis using ChIP-seq data. Motif models for PAX 2,4,6,8 and ZBTB16 were obtained from TRANSFAC (Matys et al., 2003) and PAZAR (Portales-Casamar et al., 2007), respectively. All motif models have log likelihood ratio greater than 3 relative to the background models.
(B) PAXIP1-binding sites are strongly associated with processes linked to T cell development. Top shows that terms in biological process GO are enriched for T cell differentiation and activation. GREAT (McLean et al., 2010) was used for analysis of functional gene annotations. Only terms with greater than 2-fold binomial enrichment are shown.
(C) PAXIP1 is enriched at sites occupied by H3K4me3. The normalized tags per million reads of PAXIP1 and H3K4me3 in a 50 bp window across a 4 kb region centered on the transcription starting sites (TSS) of NCBI Build 37 RefSeq genes is plotted.
(D) Correlation between alteration in H3K4me3 and direct PAXIP1-binding. For each lineage, the percent increase in PAXIP1 occupancy of promoters with more than 2-fold differential H3K4me3 (TSS +/− 5kb) was computed relative to a control set of the same size consisting of the next most relevant PAXIP1-dependent promoters with the highest increase in H3K4me3 not exceeding 2-fold.
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9. Figure 7 PAXIP1 Is Required for S1pr1 Expression
(A and B) A very few genes exhibit H3K4me3 deficit in both Paxip1−/− CD4+ SP and Paxip1−/− CD8+ SP T cell lineages. The Venn diagram shows the overlap of genes with more than three times reduction in H3K4me3 TPM in both Paxip1−/− CD4+ and CD8+ SP T cells. The numbers represent the total number of unique gene symbol in each area.
(B) List of genes with more than three times reduction in H3K4me3 TPM in both Paxip1−/− CD4+ and CD8+ SP T cells. The second and third columns show H3K4me3 reduction in Paxip1−/− CD4+ and CD8+ SP T cells with respect to WT CD4+ SP and CD8+ SP T cells, respectively.
(C) Expression of Foxo1, Klf2, and S1pr1 in sorted DP, CD4+, and CD8+ WT (black) and Paxip1−/− (red) thymoctyes measured by qRT-PCR.
(D) Surface S1PR1 expression on TCRβhiCD69lo mature thymocytes from WT (black) and Paxip1−/− (red) mice.
(E) β7-integrin and CD69 expression in CD4+ SP T cells was measured by flow cytometry using the same number of total WT (black line) and Paxip1−/− thymocytes (red line).
(F) S1PR1 overexpression decreases accumulation of mature CD4+ and CD8+ SP T cells in Paxip1 deficient thymocytes (top) and normalizes expression of maturation markers CD69, HAS, and β7-integrin in SP T cells.
Immunity  , DOI: ( /j.immuni )
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